Isolated mammalian neural stem cells, methods of making such cells

ABSTRACT

Using a novel culture approach, previously unknown populations of neural progenitor cells have been found within an adult mammalian brain. By limiting cell-cell contact, dissociated adult brain yields at least two types of cell aggregates. These aggregates or clones of stem/precursor cells can be generated from adult brain tissue with significantly long postmortem intervals. Both neurons and glia arise from stem/precursor cells of these cultures, and the cells can survive transplantation to the adult mammalian brain.

This application is the National Stage of International Application No.PCT/US98/00366 filed Jan. 7, 1998, which claims priority of, andincorporates by reference in its entirety, U.S Provisional ApplicationSer. No. 60/034,910 filed Jan. 7, 1997.

This research has been partially funded by a United States federal grantfrom the National Institutes of Health, Grant Number: NIH/NINDS1R01NS29225. The United States Government may, therefore, have certainrights to this invention.

1. FIELD OF THE INVENTION

This invention relates generally to novel mammalian brain cell types andmethods of culturing such cells. The methods of the instant invention,which utilize suspension cultures and factors that limit cell contacts,result in an amplification of the production of neural stem andprogenitor cells, and clones of such cells, from the adult mammalianbrain, including the human brain and from tissue with significant (e.g.1 day) postmortem intervals. Propagation of neural stem and progenitorcells is relevant to the large-scale production of glial and neuronalcells, and clones of such cells, as well as self-repair of the brain inneurological disease.

2. DESCRIPTION OF THE RELATED ART

Prior to the present invention, cells from numerous tissues have beendescribed that have attributes of stem or germ cells (i.e., spermatozoonor an ovum), and that are extremely well-suited for rapid self-renewal.Brain-derived stem cells have only recently been a major focus ofattention, using a variety of lineage tracing and culture methodologies.See for example, Gage et al., Ann. Rev. Neurosci., 18:159-192 (1995);Svendsen et al., Trends Neurosci., 18:465-467 (1995); Alvarez-Buylla etal., Stem Cells, 13:263-272 (1995); Weiss et al., Trends Neurosci.,19:387-393 (1996); Steindler et al., Prog. Brain Res., 108:349-363(1996); and Brustle et al., Neuron, 15:1275-1285 (1995).

Previous studies showed the presence of a dense extracellular matrix(“ECM”) on and around subependymal zone (“SEZ”) cells of the adultrodent (see, Gates et al., J. Comp. Neurol., 361:249-266 (1995); andThomas et al., Glia, 17:1-14 (1996)). ECM molecules may facilitate cellmovement and aspects of differentiation during development, and they arealso implicated in a number of neuropathological conditions.Glycoproteins such as tenascin-C (TN) and proteoglycans such as thechondroitin sulfate-containing proteoglycans (CSPG) are expressed inhigh levels in the young brain, where they seem to have a role informing glycoconjugate-rich boundaries around different functionalgroups of neurons, such as the somatosensory whisker barrel fields andstriosomes in the striatum. They are then down-regulated in later stagesof development (e.g. postnatal days 14-21) and normal adulthood, buttheir expression is enhanced in neuropathologic conditions, such astraumatic brain injury, where they are an important component of glialscar formation. In the astroglial/mesonchymal scar, they may create abarrier that inhibits the growth of neurites into the scar, although ithas been proposed that some ECM molecules may actually encourageneuritic growth under some circumstances. It has also been suggestedthat ECM molecules regulate cell proliferation, differentiation,migration, and survival through cell-cell and cell-ECM interactions.Stem cells have been described in embryonic and postnatal mouse brainand in proliferative “neurospheres” that can be harvested and culturedfrom different brain areas, including the developing subventricularzone. See, for example, Cattaneo et al., Nature 347:762 (1990); Richardset al., Proc. Nat'l Acad. Sci. (USA), 89:8591-8595 (1992); Reynolds etal., Science, 255:1707-1710 (1992); Reynolds et al., J. Neurosci.12:4565-4574 (1992); Reynolds et al., Dev. Biol., 175:1-13 (1996);Vescovi et al., Neuron, 11:951-966 (1993); Kirschenbaum et al., CerebralCortex, 6:576-589 (1994); Kirschenbaum et al., Proc Nat'l Acad. Sci.(USA), 92:210-214 (1995) Fillmore et al., Neurosci Abs., 21:1528 (1996);and Gritti et al., J. Neurosci., 16:1091-1100 (1996). Evidence fromimmunolabeling and cell birthday analyses has pointed to the existenceof such cells in the adult SEZ. See, for example, Luskin et al., Neuron,11:173-189 (1993); Menezes et al., J. Neurosci. 14:5399-5416 (1994);Levison et al., Neuron. 10:201-212 (1993); Gates et al., J. Comp.Neurol., 361:249-266 (1995), Zerlin et al., J. Neurosci. 15:7238 (1995);Thomas et al., Glia, 17:1-14 (1996); and Jankovski et al., J. Comp.Neurol., 371:376 (1996). The combination of stem/precursor cells, and adense ECM in the peri-ventricular SEZ throughout the neuraxis hasprompted the inventors of the instant invention to refer to this area asbeing the neuropoietic “Brain Marrow” (Steindler et al, Pros. Brain Res.108: 349, (1996)) since it contains elements in common withhematopoietic bone marrow.

In addition, it has recently been described that small numbers ofneurons were found to arise from precursor cells of adult human temporallobe (Kirschenbaum et al., Cerebral Cortex, 6:576-589 (1994), Laywell etal., Neurosci. Abs. 23:297 (1997)). The production of proliferatingprogenitor cells from the adult rodent brain and spinal cord has alsobeen recently described (see, Gritti et al., J. Neurosci., 16:1091-1100(1996); and Weiss et al., J. Neurosci., 16,7599-7609 (1996)). This issurprising in that with few exceptions, neuronogenesis has traditionallybeen thought to end shortly after birth in the mammalian central nervoussystem (CNS) (see, Gage et al., Ann. Rev. Neurosci. 18:159-192 (1995)).The possibility that multipotential stem cells persist in the adultbrain has implications for neuroregeneration and CNS transplantation.Accordingly, there is a need in the art for such technology. This needis met by the present invention.

The present invention discloses an advancement in the biological arts inwhich previously unknown brain stem cells are cultured and isolated. Thebrain stem cells are characterized in that the daughter cells of thebrain stem cells differentiate into neurons and glia and, therefore, areuseful in neuroregeneration cell biology, and CNS drug-effects anddrug-discovery studies. The novel method of isolating such cellscomprises culturing dissociated adult mammalian brain in conditions thataffect cell-substrate and cell-cell contacts. The cultured aggregatessurvive transplantation to the adult mammalian brain. Followingtransplantation, the daughter cells of the transplanted stem cellsdifferentiate into other cell types, including but not limited to glia,neurons, astrocytes, and oligodendrocytes, thus allowing for replacementof cells damaged by injury or disease.

Studies of the ECM molecules in the adult brain revealed the existenceof an ECM-rich pathway within which neuronal progenitor cellsproliferate and migrate. These ECM molecules play a significant role inthese events. According to the invention, the in vitro manipulation ofthese and related molecules affects cellular adhesity to other cells orsubstrates, and affects the growth of neural stem and progenitor cells,as described below.

To discourage cell-cell interactions that induce cellulardifferentiation, and thus contribute to an increased cellularproliferation of stem/progenitor cells, dissociated cells from the adultbrain were cultured in factors that interfere with protein-proteininteractions, or in gelatinous organic substances that also discouragecell contacts and allow the isolation of clonally-related colonies(spheres) of cells. There is a great expansion of the numbers of adultbrain stem and progenitor cells due to these conditions, withpotentially up to millions of neuronal and glial progenitors from smallnumbers of founder cells in less than three months. When aggregates ofprogenitor cells are plated on particular extracellular matrix moleculesubstrates in the presence of different growth factors, hormones,steroids, and other factors (see, Examples 3, and 10), theydifferentiate into neurons and glial cells. Such cells are suitable forstudies of drug-discovery and testing using clones of glial and neuronalcells as well as for cell replacement therapies in a variety of brainstructures (e.g. in the brain or spinal cord for regeneration orspace-occupation, as in spinal cord syrinx injuries, or stroke cavities,arteriovenous malformations, epileptic foci, or peripheral nerveneuromas).

Stem cells appear to make up 0.001-0.01% of an entire population ofcells in renewing or potentially renewing tissues such as bone and“brain marrow” (see, Thomas et al., Glia, 17:1-14 (1996)). Hence, anymethod that assures a large scale production of differentiated cellsfrom a small number of the most primitive stem/precursor cells isextremely useful for self-repair (autologous cell replacement) therapiesfollowing traumatic or degenerative disease. The invention harvestsbrain cells from a variety of sources including, but not limited to,rodent brain specimens, human brain biopsy, and post-mortem mammalianbrain tissue. The invention method includes isolation and amplificationof neural cells as a therapeutic self-repair approach for neurologicaldisease, particularly human disease. To accomplish the isolation andamplification, the present invention uses a novel tissue cultureapproach to maximize the isolation of stem cells, and to assure amaximal number of transplantable cells from a very small number of stemcells, thus assuring the least risk for complications from a targetedand small biopsy site. At the same time, the culture method offers thepossibility for large scale cell production from a very limited numberof extracted stem cells. Furthermore, since a more primitive type ofstem/precursor cell, than any seen prior to now, has been isolated andexpanded using the invention technology, this cell type is more amenableto central nervous system (CNS) transplantation and integration, as wellas gene transduction approaches. For example, using various vectors, theisolated cells of this invention can be transduced with genes coding fordesired functions. For example, vectors containing genes coding foradhesion molecules can be transduced into these cells to increasecellular adhesivity. This would be useful, for example, to produce gliawith increased adhesivity for cavity-filling approaches. Alternatively,the isolated cells of the present invention can be transduced with genescoding for growth factors and directing neuronal phenotype, includingneurotransmitter associated genes to produce neurons capable ofincreased neurotransmitter production. In addition, because the cells ofthe present invention are pluripotent, they are extremely well suitedfor cell-grafting approaches.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.Other objects, advantages and novel features will be readily apparent tothose skilled in the art from the following detailed description of theinvention, when considered in conjunction with the accompanyingdrawings.

The accompanying drawings, which are included to provide a furtherunderstanding of the invention, are incorporated in and constitute apart of this specification. They illustrate several embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention.

FIGS. 1A-F shows phase contrast and electron microscopic images of typeI, II and III clones. FIGS. 1A, 1C, and 1E are phase contrast images oftype I, II and III clones of cultured adult brain cells, respectively.FIGS. 1B, 1D, and 1F show type I, II and III spheres counterstained withpropidium iodide, respectively. Scale bars for FIGS. 1A-F are 40, 30,20, 30, 20, and 30 microns, respectively;

FIG. 2 depicts the types of spheres found in the culture paradigm of theinvention, and the generation conditions for the appearance andevolution of sphere types from brain;

FIGS. 3A-D shows the phase and electron microscopic images of type II (Aand B) and type III (C and D) spheres. Scale bars for FIGS. 3A-D are 10,5, 15, and 2 microns, respectively;

FIGS. 4A-J shows immunostaining of early and late type II and type IIIspheres. Scale bars for FIGS. 4A, G and J are 10 microns, FIGS. 4B and4C are 15 microns, FIGS. 4E and 4F are 30 microns, FIG. 4H is 20microns, and FIG. 4I is 100 microns;

FIGS. 5A-D shows the evolution and proliferation of type II (FIGS. 5Aand 5C), and type III (FIGS. 5B and 5D) spheres. Scale bars for FIGS. 5Aand 5B are 25 microns, and for FIGS. 5C and 5D are 10 microns;

FIGS. 6A-B shows type II and type III spheres from ROSA-26 transgenicmice. Scale bars for FIG. 6A is 50 microns, and FIG. 6B is 30 microns;and

FIGS. 7A-D shows phase and electron microscopy of a type II adult mouseand type II adult human sphere. The adult mouse sphere is approximately100 microns in diameter, while the adult human sphere is approximately200 microns in diameter.

The present invention describes methods which can be used to isolate,amplify, and grow stem/precursor cells from the mammalian brain. Bymanipulating specific aspects of cell separation and cell adhesion, theinvention methods described herein can be used to specifically isolateand culture type I, type II and type III precursor cells. It should benoted that in a recent paper by Kukekov et al. (Glia, 21:399, (1997))the type II and type III cells of the instant invention are referred toas type I and type II cells, respectively, for convenience.

Cell separation and cell adhesion can be manipulated using a variety ofcontact-limiting and contact-inhibiting factors. For example,chemical-separating agents such as mercaptoethanol, physical separatingagents such as methylcellulose, and anti-adhesives such as poly2-hydroxyethyl methacrylate are used to deter cell-cell andcell-substrate associates during the initial isolation of stem/precursorcells from the newly-dissociated brain. This allows the “purification”of these cells from mature, differentiated neurons and glia that arealso dissociated during the brain dissociation procedures. The mature,differentiated neurons and glia cannot survive these anti-adhesion,anti-cell interaction procedures. Thus, agents such as mercaptoethanolare always used in the first stage of isolation of type I and II clonesto help deter the survival of the more mature cellular elements (bydeterring their clustering). At the same time, agents such asmercaptoethanol may have certain growth-promoting actions on the singlestem/precursor cells that eventually proliferate to form these earlysphere types.

Since cell-cell and cell-substrate interactions are important forcellular differentiation, contact-inhibiting (or contact-limiting)factors as mercaptoethanol are eventually removed from the culturemedium for the evolution or differentiation of type II and type IIIspheres. The differentiation of type III spheres requires otheradditional factors, including growth factors like beta fibroblast growthfactor, epidermal growth factor, or such factors that are also containedwithin pituitary extract. Such additional factors are described in thetype III culture media discussed below (see, Example 3).

The adult mammalian brain harbors a discrete, prolific population ofprimitive stem/precursor cells that possess many of the attributes ofstem/precursor cells seen in other organs. Since these earliest (mostprimitive) cells do not readily attach to culture substrata, and requirecontact-limiting factors for amplification, they may have beenoverlooked in previous culture studies of adult brain stem andprogenitor cells. Under certain culture conditions, these cells evolveinto many different classes of cells, including progenitors that giverise to neurons and different types of glia. In this way, they possessmany of the cytological characteristics of hematopoietic stem/precursorcells that can give rise to different types of blood progenitor cells.These brain stem/progenitor cells may be produced on a large scale andcan be genetically altered using a variety of transfection methods.

The pluripotential brain cells of the instant invention can be directedto particular neuronal or glial lineages. Thus, the culturing methods ofthe instant invention can be used to produce large numbers ofclonally-related cells of a specific cell type. The methods of producingsuch large populations of cells as well as the cells themselves arespecifically useful in replacing cells that are lost due to diseaseprocesses. For example, the production of large quantities of specificglial cells by the methods of the present invention and subsequenttransplantation of these glia into the brains of multiple sclerosispatients, would be particularly beneficial to such patients. Similarly,neurons generated from the stem/precursor cells of the instantinvention, transplantation of these neurons, and the integration of theactivities of the replaced cells into established brain circuitries isparticularly useful in brains where neuronal cell loss has occurred.Cells generated using the methods of the instant invention can also beused in the brain or spinal cord for regeneration or space-occupation,as in spinal cord syrinx injuries, or stroke cavities, arteriovenousmalformations, epileptic foci, or peripheral nerve neuromas to fillcavities. Large or small scale production of the cells using theinvention methods are also useful for drug-discovery and testing.

The culture paradigms described below yield three morphologically andantigenically distinct populations of cellular aggregates, types I, IIand III. These populations of cellular aggregates are termed “clones”,each clone having distinct characteristics (see, FIG. 1). As shown inFIG. 1A, clones of type I appear as phase-bright, very small densebodies. It is not possible to discern individual cells within the clonesusing phase microscopy. However, when the cells are counterstained withpropidium iodide (PI) (see, FIG. 1B), type I clones exhibit areas ofvery small, punctate staining interspersed with regions that lackstaining. Type I clones do not appear to attach to either plastic orlaminin-coated substrates, and cells of individual clones are notseparable by trypsinization. Furthermore, type I clones areimmunonegative for all of the cell-specific markers tested so far.

Type II clones from adult mouse and human brain dissociations,spontaneously appear in suspension cultures (see FIGS. 1C, 1D, 3A, and3B), containing at least one contact-limiting or contact-inhibitingfactor. Some exemplary contact limiting factors include, but are notlimited to, mercaptoethanol, poly 2-hydroxyethyl methacrylate, andmethylcellulose. Mercaptoethanol functions as a chemical-separatingagent as it breaks disulfide linkages of proteins which are involved incell-cell and cell-substrate interactions. Methylcellulose functions asa physical separator; it is a viscous bioorganic solution which, by itsviscous nature, limits contacts between cells and between cells and asubstrate, and allows clonal analyses. Poly 2-hydroxyethyl methacrylatefunctions as an anti-adhesive for substrate coating. This prevents cellcontact with adhesive surfaces and also prevents differentiation. Othercompounds or procedures which function to limit or inhibit cell-cell andcell-substrate interactions can also be used with this invention.Culturing as a suspension also deters cell-cell and cell-substratecontacts with higher yields of spheres, but culturing in methylcelluloseagain, allows clonal analyses.

In contrast to type I clones, type II clones are phase-dark, sphericalbodies that become larger with time (FIGS. 1C and 3A). ElectronMicroscopy (EM) revealed that type II clones consist of rings of small,tightly apposed cells that surround a core of flocculent, non-cellularmaterial having many characteristics of extracellular matrix (FIGS. 1Dand 3B). The type II cell has many organelles, including endoplasmicreticulum, Golgi apparatus, dense bodies, and mitochondria. The closedarrow in FIG. 1C points to a multisphere aggregate (as shown bypropidium iodide counterstaining, a DNA stain for cell nuclei, in FIG.1D). The closed arrow in FIG. 1E points to a type II sphere residingnear a type III sphere, which is marked by an open arrow.

Type II clones do not attach to either plastic or laminin-coatedsubstrates. Immediately after they appear in culture, type II clones areimmunonegative for cell-specific markers, including GFAP (glialfibrillary acidic protein, which labels more mature and reactiveastrocytes), nestin (RAT401, which stains neurepithelial stem cells aswell as radial glia), and TuJ1 (which stains class III β-tubulin that isexpressed in recently postmitotic (committed) as well as mature neuronsin the CNS). However, after approximately ten days to two weeks invitro, some cells of type II clones (late type II, or early type III)become immunopositive for nestin, but remain immunonegative for GFAP andTuJ1 (see, FIG. 4). If the factors that inhibit cell contact are removedfrom the medium, provided the type II clones are not kept in thecontact-limiting factor for more than two weeks, type II clones willconvert to type III clones.

In contrast to type II clones, type III clones, when plated on plasticor laminin-coated substrates, attach readily, and produce a number ofprocess-bearing cells that migrate away from the sphere to from a singlelayer of cells. Type III cells are immunopositive for a variety ofcell-specific markers, including GFAP, nestin, L1 (a marker of anadhesion molecules that is on the surface of neurons and theirprocesses), and TuJ1 (see, FIG. 4). Some cells exhibit very light,punctate staining using an antibody to the 04 antigen (marker ofoligodendrocytes).

In addition to the type I, type II and type III clones, the primarycultures of the instant invention also appear to initially containdifferentiated neurons and glia. However, in the presence ofcontact-inhibiting factors, neurons and astrocytes were notimmunologically detected after 1 week; they presumably cannot surviveunder these culture conditions.

The culture paradigm of the invention, and the generation conditions forthe appearance and evolution of sphere types from a brain are depictedin FIG. 2. Both type II and type III stem/precursor cells are generatedfrom a dissociated adult mammalian brain. Type II cells appear 2-3 weeksafter mercaptoethanol, or other cell contact limiting factor, is addedto the culture medium. When mercaptoethanol, or other contact-limitingfactor, is subsequently removed from the medium after the appearance oftype II spheres, the type II spheres increase in size and, after anadditional 10-14 days, convert to type III spheres. In addition todeveloping from type II clones after the addition and subsequent removalof a contact limiting factor, type III clones can also appearspontaneously in suspension cultures grown in the absence of contactlimiting factors.

FIG. 3 shows the phase and electron microscopic images of type II (A andB) and III (C and D) spheres. After approximately 30 days in vitro, typeII clones take on characteristics of type III clones. The size of thetype II clones increase dramatically, and their color changes fromphase-dark (FIGS. 1C and 3A) to phase-bright (FIGS. 1E and 3C). Inaddition, large cells appear at the edges of the type III clones.

FIG. 3C shows type III spheres which are generally larger andphase-brighter than type II spheres. The open arrow points to a latetype II or early type III sphere, and the closed arrow points to a largecellular protrusion on the periphery of a late type III sphere whichappears brighter than the early type III, and has a more irregularborder due to many protrusions. FIG. 3D shows cells of type III sphereswhich look more mature than type II cells. The open arrow points to alarge cell, and the closed arrow marks a smaller cell with a darkernucleus.

Type III spheres will continue to increase in size and begin to displaycellular heterogeneity when grown in suspension culture. Eventually,cells within suspended type III spheres begin to express markers ofdifferentiation. Such cells can eventually become neurons and glia whenplated onto favorable substrata such as laminin/poly ornithine coatedsurfaces, polylysine, or plastic, or other matrix molecules, ortransplanted into a host brain.

FIG. 4 shows the immunostaining of early and late type II and IIIspheres illustrating that the various characteristics of the type II andtype III spheres can be readily distinguished from their immunostainingprofiles and their phase-contrast, and EM images. Early type II spheresare shown in FIG. 4A. The open arrows point to two type II spheres thatabut each other. These spheres are negative for the putative stem cellintermediate filament protein, nestin. A more mature type II sphere thatis immunopositive for nestin is shown in FIG. 4B. FIG. 4C shows an earlytype III sphere immunopositive for nestin, with some unlabeled cellsalso apparent. FIG. 4D shows a phase contrast image of a late type IIIsphere (open arrow) showing processes after attaching to plastic. FIGS.4E and 4F show a type III sphere, with cells beginning to disperse afterattaching to plastic. This sphere is double immunostained for GFAP (FIG.4E) and β-III tubulin (FIG. 4F). FIG. 4G depicts astrocytes and showsthat dispersed cells of type III spheres are immunopositive for nestin.FIG. 4H shows astrocytes from dispersed type III spheres also stain forGFAP. The open arrow points to a morphologically different astrocytethan the large cell on the right. FIG. 4I shows cells that are dispersedfrom an attached type III sphere and which are immunopositive for L1.The arrow points to a long L1-positive process. FIG. 4J shows a singlecell immunopositive for β-tubulin, counterstained with propidium iodide(orange), after attachment of a type III sphere. Thus, type II and typeIII spheres are easily distinguishable from each other, not only by thedifferences in phase contrast, and EM images, but also by their distinctimmunostaining profiles.

The evolution and proliferation of a type II sphere differentiating intoa type III sphere can be seen in FIG. 5. A single late type II sphere(arrow in FIG. 5A) was followed in suspension culture with phasemicroscopy. After 10 days, this type II sphere had increased in size andaltered its morphology to become the type III sphere shown in FIG. 5B.The open arrowhead points to a cellular protrusion on the edge of thesphere typical of the type III sphere. Cellular debris around the typeII sphere in FIG. 5A is absent from the type III sphere in FIG. 5B dueto movement during feeding. Heterogeneous cultures of spheres exposed toa 20 hour pulse of BrdU reveal proliferative cells in both type II (FIG.5C) and type III (FIG. 5D) spheres. Notice that both the small sphere inFIG. 5C and the larger sphere in FIG. 5D contain labeled nuclei with avariety of sizes. This indicates that there may be different progenitorcells types proliferating in the spheres; some progenitor cell typesgiving rise to neurons, some giving rise to glia.

Removal of the contact inhibiting factors will allow differentiation tooccur. In addition, the type II and type III growth media containinggrowth factors such as basic fibroblast growth factor (bFGF), orepidermal growth factor (EGF) encourages differentiation. Other growthfactors such as brain-derived neurotrophic factor (BDNF), glial derivedneurotrphic factor (GDNF), NT3, and ciliary neurotrophic factor (CNTF)may also encourage differentiation of the stem/precursor cells.

It should also be noted that the flat, spread appearance of the type IIIsphere in FIG. 5D is probably due to this sample being taken from a 96hour culture as opposed to the 48 hours for the type II sphere, and typeIII spheres have a tendency to spread and migrate when attached. Thelack of spreading of the type II sphere may also account for the higherlevel of background staining.

Type II and type III clones were also generated from ROSA-26 mice, astrain that expresses the β-galactosidase transgene in all cell types,to perform transplant experiments. ROSA-26 transgenic mice arecommercially available from the Jackson Laboratories, Bar Harbor, Me.The results show that following transplantation into adult mouse brain,type II and type III clones can survive and differentiate. (FIG. 6).When these β-galactosidase positive type II and type III clones weretransplanted to the striatum of adult ICR mice, small and large X-galpositive cells were found to survive up to two weeks. Moreover,immunocytochemistry with GFAP revealed the presence of labeledastrocytes and non-labeled larger cells (presumably neurons). FIG. 6Ashows a β-galactosidase positive late type III sphere (large openarrow), and unlabeled early (right open arrowhead) and late (left openarrowhead) type II spheres. The filled arrow points to unidentifiedlabeled elements out of the plane of focus. FIG. 6B shows the combinedbright field/fluorescence image of a transplant of type II and IIIspheres into the striatum of an adult ICR mouse. Surviving astrocytesmarked with a filled arrow are counterstained for GFAP. LargeGFAP-negative, β-galactosidase-positive cells, presumed to be neurons(open arrow), are also seen. Other transplanted cells can be seen withinthis host brain structure as well. Immunofluorescence is yellow; theβ-galactosidase product is blue-green. These results support theconclusion that the novel stem/precursor cells are useful inregeneration following neurological cell damage.

Type II and type III clones were also generated from the adult humanbrain, and from dead animals with long postmortem intervals when theanimals were kept at 4° C. (see, FIG. 7). FIG. 7 shows phase andelectron microscopic images of type II adult mouse and human spheres.FIG. 7A shows the phase microscopic image of a type II adult mousesphere that looks similar to a sphere with a postmortem interval of 0hours, while FIG. 7B shows the phase microscopy of a type II sphere froman adult mouse with a postmortem interval of sixteen hours. FIG. 7Cshows the electron microscopic image of a type II sphere from an adulttransgenic mouse (tenascin glycoprotein knockout mouse). A complexcellular aggregate can be seen. This sphere is approximately 100 micronsin diameter. FIG. 7D shows the electron microscopy of an adult humansphere (as shown in the inset, a phase microscopic image of a type IIsphere). Similar to the electron microscopy of a type II sphere from anadult transgenic mouse (FIG. 7C), this human sphere (FIG. 7D) also showsa complex cellular aggregate. This human sphere is approximately 200microns in diameter.

The different types of clones observed in the cultures described aboveand in the experiments described below, represent a continuum of cellproliferation and differentiation, with the existence of both early andlate type II clones, which can be compact or loose in appearance, basedon cell packing density, that eventually differentiate into type IIIclusters (see FIGS. 1 and 3). The potential for numerous, undefinedhematopoietic stem cells still exists (see, for example, Larochelle etal. Nature Med., 2:1329-1337 (1996)). The identification andunderstanding of neuropoietic stem and progenitor cells based on acombination of morphology, gene marking, and unique biochemicalfeatures, such as that described herein, ensure reliability in theresults. The use of just one feature as an identification tool canoccur, although it makes the recognition of the specific stem cell typerather tenuous.

While the inventors do not wish to be bound by any particular theory,neuropoiesis in the adult brain is probably a rather limited event,based on current knowledge of hematopoiesis and the presence of stemcells in other tissues, as well as the apparent existence of a quiescentpopulation of so-called stem cells in the SEZ (see, Potten et al.Development, 110:1001-1020 (1990); and Morshead et al., Neuron13:1071-1082 (1994)). For this reason, methods that amplify the ex vivoproduction of these cells, as described herein, are useful to generatelarge numbers of such cells for classification and transplantation.Furthermore, the methods of the instant invention can be applied toharvesting the small number of stem/precursor cells from adult brains,particularly adult brains with significantly long postmortem intervals(e.g. 1 day), allowing the “banking” of these cells for future studiesand cell-replacement therapies.

Also, novel approaches as described here, that uncover either novelstem/precursor cells or aspects of their growth and differentiation thatlead to classification of stages of adult brain neuropoiesis are alsouseful. The described methods allow this process with the production ofthe most primitive stem/precursor cells, and facilitate the generationand analyses of a continuum of developing and differentiating brainstem/precursor cells.

The culture conditions of the present invention involve limitingcell-cell and cell-substrate interactions leading to the enhancedproduction of spheres of type II and III cells. Since previous studieshave only shown a dense extracellular matrix within the SEZ in vivo(see, for example, Gates et al., J. Comp. Neurol., 361:249-266 (1995);and Thomas et al., Glia. 17:1-14 (1996)), and since ECM molecules havebeen reported to affect the proliferation and differentiation ofhematopoietic stem cells (see, for example, Klein et al., J. Cell Biol.,123:1027-1035; and Yoder et al., Exp. Hematol., 23:961-967 (1995)), andwhile the inventors do not wish to be bound by any particular theory, itis presumed, that the methods of the present invention affect theactions of the ECM molecules, adhesion proteins, growth factors andtheir interactions. Preliminary studies using immunocytochemistry andthe reverse transcriptase polymerase chain reaction (rtPCR) suggest thatsphere cells express transcripts as well as protein of particular ECMmolecules (e.g. tenascin).

As described above, the appearance of two types of proliferating cellsthat form neurosphere-like aggregates from dissociated adult brain,termed type II, and III clones, were consistently observed. While thetype III clones in the culture paradigm of the invention most likelyrepresent neural progenitor cells that have previously been described inthe aforementioned papers (see, for example, Reynolds et al., J.Neurosci., 12:4565-4574 (1992); Reynolds et al., Dev. Biol., 175:1-13(1996); and Weiss et al., J. Neurosci., 16:7599-7609 (1996), all citedabove), it has been heretofore unrecognized that such type III spheresare derived from the type II precursors of the instant invention. Byisolating and amplifying type II spheres in specific culture conditions,type III spheres can be obtained. In addition, prior to the disclosureof the methods of the instant invention, type II and type III sphereshave not been previously obtained from post-mortem brain specimens.

In addition, prior art stem cells are not as primitive as the type IIstem cells of the present invention as evidenced by the fact that theprior art stem cells are all nestin-positive. The type II clones of theinstant invention represent a truly unique, ontogenetically earlier formof stem/progenitor than those previously described. This conclusion issupported by data showing that the type II clones are first initiallynestin-negative, followed by a progression through a nestin-positivestate to become a type III clone, and finally differentiation intoneurons or glia (see, FIGS. 2-4).

The following examples are offered by way of illustration and are notintended to limit the scope of the claimed invention.

EXAMPLE 1 The Production of Type I Clones

Adult ICR or transgenic or mutant mice, or biopsy specimens from humantemporal lobe (for epilepsy surgery), or brain specimens withsignificant (i.e. at least one day) postmortem intervals, were used astissue sources for dissociations. The brains were dissociated andcultured follows. Extracted brain tissues were minced with a razor bladeand washed in a mixture of ice-cold DMEM (Dulbecco's modified Eagle'smedium, commercially available from a variety of vendors) and anyantibiotic-antimycotic product such as Sigma Chemical Co. Catalogue#A5955 (100×). (Such antibiotic-antimycotic products are alsocommercially available from Gibco Brl, Grand Island, N.Y.). Brain pieceswere transferred to a beaker containing 0.25% trypsin and EDTA(ethylenediaminetetraacetic acid) and mixed on a magnetic stir-plate for15 minutes, triturated with a plastic pipette, filtered through sterilegauze, and collected in a 15 ml tube and centrifuged for 5 minutes at1200 rpm. Cells were resuspended in DMEM/F12 medium+N1 supplement (astandard tissue culture medium available from a variety of vendors) plus5% FBS (fetal bovine serum) and grown in suspension cultures by platingat high density on a non-adhesive substrate (tissue culture plasticcoated with poly 2-hydroxyethyl methacrylate; Sigma Chemicals). Cellswere fed every 3-4 days by centrifugation and resuspension in freshmedium.

The basic media for culturing type I cells comprises the followingingredients: Insulin (5 μg/mL), putrescine (100 μM), progesterone (20μM), sodium selenite (30 μM), pituitary extract (20 μg/mL), transferrin(100 μg/mL), and 5% fetal calf serum (FCS) in DMEM/F12 media.

Type I cells only appear in suspension cultures containing anon-adhesive substrate such as poly 2-hydroxyethyl methacrylate. Sometype II cells are also present in these cultures.

EXAMPLE 2 The Production of Type II Clones

Type II clones, similar to type I clones, were obtained from adult ICR,transgenic or mutant mice, or biopsy specimens from human temporal lobe(for epilepsy surgery). In addition, Type II clones were also generatedfrom the adult human brain, and from dead animals with long post mortemintervals when the animals were kept at 4° C.

The brains were dissociated and cultured as previously described for thegeneration of type I clones. Briefly, extracted brain tissues wereminced, washed, and transferred to a beaker containing 0.25% trypsin andEDTA. After being mixed on a magnetic stir-plate for 15 minutes, theculture was triturated with a plastic pipette, filtered through sterilegauze, centrifuged, and resuspended in DMEM/F12 medium+N1 supplement,plus 5% FBS, plus 20 μg/mL pituitary extract (from Gibco) and grown insuspension cultures by plating at high density on a non-adhesivesubstrate.

Cells were plated and fed as described above for the type I cells.However, the basic media described above (comprising insulin (5 μg/mL),putrescine (100 μM), progesterone (20 μM), sodium selenite (30 μM),pituitary extract (20 μg/mL),transferrin (100 μg/mL), and 5% fetal calfserum (FCS) in DMEM/F12 media) also contained 10 ng/mL basic fibroblastgrowth factor (bFGF), and 10 ng/mL epidermal growth factor (EGF).Importantly, the culture media additionally contained 100 μMmercaptoethanol as a contact-limiting factor that reduces disulfidebonds (See, E. C. Herrington, Biochem. Pharmacol., 35:1359-1364 (1986).)Cultures contained dense debris for 10-14 days. Mercaptoethanol was thenremoved from the medium after 10-14 days. Clones of type II were presentin these cultures. Some type III clones were also present.

EXAMPLE 3 The Production of Type III Clones

Similar to the type I and type II clones, Type III clones were obtainedfrom adult ICR, transgenic or mutant mice, or biopsy specimens fromhuman temporal lobe, or from the adult human brain, or from dead animalswith long post mortem intervals when the animals were kept at 4° C. Thebrain source was dissociated as described in Examples 1 and 2 above, andthe cells were grown in the suspension culture described above eitherwithout contact inhibiting factors, or, more often with a contactinhibiting factor such as mercaptoethanol. The basic media for culturingtype III cells was the same as that used for culturing type II cells.Namely, the media comprised insulin (5 μg/mL), putrescine (100 μM),progesterone (20 μM), sodium selenite (30 μM), pituitary extract (20μg/mL), transferrin (100 μg/mL), 10 ng/mL basic fibroblast growth factor(bFGF), 10 ng/mL epidermal growth factor (EGF), and 5% fetal calf serum(FCS) in DMEM/F12 media. Cells were fed every 3-4 days by centrifugationand resuspension in fresh medium. After removal of the contact limitingfactor, both type II and Type III clones were apparent after 5-7 days.The type II clones eventually evolved into type III clones uponcontinued culturing in the absence of contact limiting factors.

Simply removing the contact inhibiting factors encouragesdifferentiation by encouraging cell-cell contact. However,differentiation of type III clones into neurons or glia is alsoencouraged by other additional factors, including the growth factorslike β-fibroblast growth factor, epidermal growth factor, or factorsthat are contained within pituitary extract present in the basic typeIII culture media. Other growth factors such as brain-derivedneurotrophic factor (BDNF), glial derived neurotrphic factor (GDNF),NT3, and ciliary neurotrophic factor (CNTF) may also encouragedifferentiation of the stem/precursor cells.

The following chart summarizes the various methods to obtain thedifferent stem/precursor cell types of the instant invention:

Steps Type I clones Type II clones Type III clones Braindissociation + + + + Grow in suspension + + + + Culture Add contactinhibiting + + + factor (for ≦ 2 weeks) Remove contact in- + + hibitingfactor Culture or plate on + + plastic/laminin coated substrate

EXAMPLE 4 Fixing of Cultures for Staining or Antibody Testing

Cultures are fixed in one of two ways depending upon the cultivationparadigm. Adherent clones (cultivated on plastic or laminin-coatedplastic) are washed three times with room temperature Dulbecco's PBS(phosphate buffered saline) and then fixed either with 10% acetic acidin pure ethanol at −20° C., or ice cold 4% paraformaldehyde in PBS.After 3/4 to 1 hour, the fixative was removed and the cultures werewashed three times with PBS.

Clones cultivated as suspension cultures were collected in a 15 mlplastic tube and centrifuged to form a pellet. Culture medium wasaspirated and fixative (described above) was added to the pellet. Cloneswere then triturated and kept in fixative for 3/4 to 1 hour, after whichtime the cells were again centrifuged and washed in PBS. Finally, thepellet was resuspended in a small volume of fresh PBS, and smallaliquots of cells were placed on polylysine-coated coverslips andallowed to dry before the application of cellular stains or antibodies.

EXAMPLE 5 Preparation of Cultures for Ultrastructural Analysis

Clones grown in suspension cultures were prepared for ultrastructuralanalysis by fixation in sodium cacocodylate buffer comprising 2%glutaraldehyde, 2% paraformaldehyde, 0.5% acroline, and 5% sucrose. Thefixative was warmed to 37° C. and gradually added to cultures until thefixative-to-medium ratio was 1:1. Cells were then collected in 15 mltubes and centrifuged to form a pellet which was then covered with 100%fixative for several hours before processing with standard embedding,staining, and sectioning protocols. Samples were viewed on a JEOL 2000electron microscope.

EM of type II clones revealed rings of small, tightly apposed cells thatoften surround a core of flocculent, non-cellular material (see, FIG.3B) having many of the characteristics of extracellular matrix. The typeII cell has many organelles, including endoplasmic reticulum, Golgiapparatus, dense bodies, and mitochondria.

EM of type III clones (see, FIG. 3D) revealed cells that appear to bemore differentiated than type II cells, as their cytoplasm is less densethan type IIs and their organelles appear to be more developed. FIG. 7shows EM of a clone from a transgenic mouse (tenascin knockout mouse)and a sphere (clone) from an adult human temporal lobectomy specimen.

EXAMPLE 6 Culturing of Cells in Methylcellulose for Observation ofSingle Clones

Methylcellulose (StemCell Technologies, Vancouver, B.C.) was dissolvedin medium (o a concentration of 1.6%, and Dulbecco/F12 +N1 supplementmedium was added, with an equal volume of brain cells suspension, to afinal concentration of 0.8% methylcellulose (see, Worton et al., J. CellPhysiol., 74;171-182 (1969)). Cells were fed every 2-3 days by theaddition of small aliquots of medium without methylcellulose. Singleclones were followed over time (see, FIG. 5), and observed to increasein size indicating cell proliferation and growth.

EXAMPLE 7 Testing Clones for Reactivity to Cell Markers

Standard immunofluorescence techniques were used with antibodies topolyclonal GFAP (Immunon), monoclonal β-tubulin (Sigma), polyclonal L1(gift of Professor Melita Schachner), monoclonal nestin (DevelopmentalHybridoma Bank), and 04 (Chemicon). Cells were also labeled withpropidium iodide (Sigma).

Type II clones are immunonegative for cell-specific markers, includingGFAP, nestin, and TuJ1. These are considered early type II clones.However, after approximately 10 days in vitro, some cells of type IIclones become immunopositive for nestin but remain immunonegative forGFAP and TuJ1. Type III clones exhibit cell phenotype markers of moredifferentiated cells being immunopositive for nestin, GFAP, L1, andTuJ1. This staining pattern shows the continuum of evolution of type IIclones from early (immunonegative) to late (selectively immunopositive)and eventually to type III clones (fully immunopositive) (see, FIG. 5).

Other techniques can be used to distinguish type II and type III clones.For example, rtPCR can be used to reveal genes that are differentbetween type II and type III clones. Not only will this furthercharacterize the type II and type III clones, but it will also revealnovel genes in each clone type, as well as identify genes involved indifferent developmental stages of neuronal precursor cells.

EXAMPLE 8 Using Brains from Dead Mammals as a Source of Stem/PrecursorCells

Using exactly the same procedures as outlined for generating type I, IIand III spheres from acutely dissociated brain tissue, it is alsopossible to generate type I, II and III clones from brain tissue ofanimals with significantly long postmortem intervals (FIG. 7B). Thusfar, dissociated brain from adult mice with postmortem intervals from16-24 hours have yielded normal type I, type II, and type III spheres.Pilot studies have indicated that it might be possible to harvest braintissue (if stored at 4° C.) from animals or human cadavers withpostmortem intervals of up to 2-5 days. These findings have implicationsfor “banking” of mature human brain tissue specimens for experimentationas well as transplantation for traumatic injuries and neurologicaldisease.

EXAMPLE 9 Light and Electron Microscopy of Clones from Adult Human Brain

Human brain also generates type II, and III clones, as described for theadult rodent. Dissociating biopsy specimens from the adult humantemporal lobe yields type II, and III spheres using the same culturemethods as described for isolating type II and type II spheres fromrodent brain tissue. In addition, similar to the adult rodent, humanbrain may also generate type I clones. FIG. 7D shows human spheres atthe light and electron microscopic level that have many of the samecytological feature as described in rodent spheres.

EXAMPLE 10 Transplantation (Grafting) of Type I or II Clones fromROSA-26 Adult Mice

Type II or III clones, generated as described in Examples 2 and 3 (see,above) from adult ROSA-26 transgenic mice (see, Friedrich et al., GenesDev., 5:1513-1523 (1991)), were aspirated, with media, into a Hamiltonmicrosyringe with an attached 31 gauge needle using a videostereomicroscope set-up. 1 μl was slowly injected into the adult ICRmouse striatum. For stereotaxic coordinates, as well as details for thehistochemical detection of β-galactosidase activity, see Gates et al.,Neuroscience, 74:579-597 (1996)). Type II and III clones were also foundto exhibit X-gal labeling in vitro (see, FIG. 6). Survival times of 7-14days were observed thus far, with a 10-day survival shown in FIG. 6. Itis possible that the transplants can survive longer times, simplycarrying the experiment out for a longer time point will determine that.Preliminary studies also indicate that grafted type II and III spheresfrom adult human brains give rise to cells that survive in the maturebrains of immunocompromised mice.

In addition, the transplants can differentiate into different types ofresident neuronal populations. For example, some of the transplantedspheres from β-galactosidase-positive mice differentiate into astrocytesin the host ICR (non-β-galactosidase-positive brain); some transplantedspheres differentiate into neurons. Immunocytochemistry with GFAPrevealed the presence of labeled astrocytes and non-labeled larger cellsthat are presumed to be neurons. Furthermore, in vitro cultures usingfeeder cells from different parts of the body (i.e. endothelial cells,lung endothelial cells, or kidney cells), change the gross morphology ofthe spheres generated. Both of these in vitro, and in vivo experimentssuggest that the environment into which the spheres are transplantedcontributes to the type of cells into which they will differentiate. Itmay therefore be possible to direct the phenotype of the precursor/stemcells using cell feeder layers from specific tissues, as well as othermolecular priming approaches, and genetic manipulations (e.g.transfections, or viral infections).

The transplant studies described above were performed in “normal” braincircuitries. Such studies indicate that type II, and III spheres giverise to glia and neuronal cells that can survive the grafting procedure.Similar experiments can be done in compromised circuitries to determinewhether or not compromised circuitries result in any lineage restrictionor allow further differentiation.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods of the presentinvention without departing from the spirit or scope of the invention.Thus, it is intended that the present invention cover the modificationsand variations of this invention provided they come within the scope ofthe appended claims and their equivalents.

We claim:
 1. A method for obtaining a purified population of primitivehuman brain stem cells, comprising culturing dissociated human braincells on a non-adhesive substrate in suspension culture supplementedwith fetal bovine serum and methyl cellulose, where culturing underconditions that inhibit cell-cell and cell-substrate interactionsresults in a substantially homogeneous population of pluripotent brainstem cells that are immunonegative for glial fibrillary protein, nestinand TuJ1 and are free from mature, differentiated neurons and glia.